CN109022355B - Culture method for improving differentiation of mesenchymal stem cells into chondrocytes - Google Patents

Culture method for improving differentiation of mesenchymal stem cells into chondrocytes Download PDF

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CN109022355B
CN109022355B CN201811040113.9A CN201811040113A CN109022355B CN 109022355 B CN109022355 B CN 109022355B CN 201811040113 A CN201811040113 A CN 201811040113A CN 109022355 B CN109022355 B CN 109022355B
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chondrocytes
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stem cells
mesenchymal stem
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CN109022355A (en
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马亚东
黄宗堂
曹娜
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Fengzekang Bio Medicine Shenzhen Co ltd
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Abstract

The invention belongs to the technical field of stem cells, and particularly relates to a culture method for improving differentiation from mesenchymal stem cells to chondrocytes, which comprises the steps of (1) extracting chondrocytes, and inoculating the chondrocytes into a cell culture solution for culture; (2) extracting mesenchymal stem cells, and inoculating the mesenchymal stem cells into an inserted cell culture dish; (3) putting the inserted cell culture dish in the step (2) and the chondrocytes cultured in the step (1) into a cell induced differentiation culture medium for culturing together; the cell induced differentiation culture medium contains human bone morphogenetic protein 2 and growth hormone HGH. The invention cultures the cartilage cells and the mesenchymal stem cells in the induction culture medium at the same time, and the two cells simulate the in vivo environment under the induction of the culture medium, thereby achieving the synergistic effect, improving the proliferation rate of the cartilage cells and simultaneously improving the expression rate and the expression quantity of the collagen of the cells after induced differentiation.

Description

Culture method for improving differentiation of mesenchymal stem cells into chondrocytes
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a culture method for improving differentiation of mesenchymal stem cells into chondrocytes.
Background
Articular cartilage is mainly a non-vascular tissue composed of chondrocytes and extracellular matrix, and nutrients are absorbed mainly by joint movement and extrusion, so that the cartilage has weak self-repair capability after being damaged, and repair and transplantation after being damaged are one of the difficult problems in the medical field. The traditional method for repairing cartilage tissue adopts autologous chondrocytes, the method must cut off articular cartilage in a non-load bearing area under an arthroscope and amplify the chondrocytes in the articular cartilage, and the operation has the unique advantage of no antigenicity but causes great damage to the body. In addition, most cartilage tissues are matrixes, the number of chondrocytes is small, the chondrocytes are difficult to separate, the number of in vitro passages is limited, and the chondrocytes are generally cultured for 3-4 generations at most, so that a large number of cells are difficult to obtain.
In order to culture and expand chondrocytes in vitro, various chondrocyte media for culturing chondrocytes have emerged. For example, autologous serum is added, but the dedifferentiation tendency of chondrocytes is serious, and the efficiency after induction is low.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a culture method for improving the differentiation of mesenchymal stem cells into chondrocytes.
In order to achieve the above purpose, the technical solution of the embodiment of the present invention is as follows:
a culture method for improving differentiation of mesenchymal stem cells to chondrocytes comprises the following steps:
(1) extracting chondrocytes, and inoculating the chondrocytes into a cell culture solution for culture;
(2) extracting mesenchymal stem cells, and inoculating the mesenchymal stem cells into an inserted cell culture dish;
(3) putting the inserted cell culture dish in the step (2) and the chondrocytes cultured in the step (1) into a cell induced differentiation culture medium for culturing together; the cell induced differentiation culture medium contains 2 x 10-7～5×10- 7mol/L human bone morphogenetic protein 2 and 4X 10-7～6×10-7mol/L growth hormone HGH.
Wherein the cell induction differentiation culture medium takes a high-glucose DMEM culture solution as a basic culture medium, and also comprises the following components:
vitamin C with concentration of 30-180 μ g/ml;
basic fibroblast growth factor (bFGF) with the concentration of 25-80 ng/ml;
transforming growth factor TGF-alpha with the concentration of 20-30 ng/ml;
transforming growth factor TGF-beta with a concentration of 10-35 ng/ml;
insulin at a concentration of 5-25 μ g/ml;
insulin growth factor IGF-1 at a concentration of 2-25 ng/ml;
bone morphogenetic protein BMP with concentration of 1-10 ng/ml;
fetal calf serum or autologous serum with the concentration of 5-25 v/v%;
dexamethasone with the concentration of 50-100 nmol/L;
sodium pyruvate with a concentration of 1-2 w%;
interleukin IL-1 at a concentration of 5-15 ng/ml.
Further, step (a)1) The extracted chondrocytes are one or two of primary cultured or subcultured chondrocytes. The inoculation amount of the chondrocytes is 0.3 multiplied by 104～0.8×104Per cm2. The chondrocytes are cultured in a cell culture solution for 1-2 days, wherein the cell culture solution is the chondrocyte culture solution.
In the step (2), the extracted mesenchymal stem cells are one or two of primary culture mesenchymal stem cells or subculture mesenchymal stem cells. The inoculation amount of the mesenchymal stem cells is 3 multiplied by 104～5×104Per cm2。
In the step (3), the culture is carried out in an incubator under the conditions of 37 ℃ and CO25 percent, pH value of 7.2-7.4, and sterile constant-temperature culture.
The method is the method, but through a plurality of experiments and summarization of the inventor, in order to achieve better effect of cell induced differentiation culture, the method can also comprise tanshinone with the concentration of 1-3mg/L, safflower powder with the concentration of 0.5-1.0mg/L, total achyranthes bidentata saponin with the concentration of 1.0-2.5mg/L and L-glutamine with the concentration of 2-3 v/v%.
On the basis, the tanshinone is a fat-soluble phenanthrenequinone compound which is extracted from traditional Chinese medicine Salvia miltiorrhiza (root of Salvia miltiorrhizae Bunge in Labiatae) and has an antibacterial effect, and the monomer has an antibacterial effect and also has anti-inflammatory and cooling effects. The total tanshinone has the effects of resisting bacteria, diminishing inflammation, promoting blood circulation to disperse blood clots, promoting wound healing, etc. and has obvious antibacterial effect in cell culture process.
The culture method for improving the differentiation of the mesenchymal stem cells into the chondrocytes, provided by the invention, is characterized in that the chondrocytes and the mesenchymal stem cells are cultured simultaneously in an induction culture medium, and the two cells simulate the in-vivo environment under the induction of the culture medium, so that the synergistic effect is achieved, the proliferation rate of the chondrocytes is improved, and the expression rate and the expression quantity of collagen of the induced and differentiated cells are improved.
Detailed Description
The specific technical scheme of the invention is described by combining the embodiment.
Example 1
A culture method for improving differentiation of mesenchymal stem cells to chondrocytes comprises the following steps:
(1) extracting chondrocytes, and inoculating the chondrocytes into a cell culture solution for culture; the extracted chondrocytes are primary cultured chondrocytes. The inoculation amount of the chondrocytes is 0.6 multiplied by 104～0.8×104Per cm2. The chondrocytes are cultured in a cell culture solution for 1 day, wherein the cell culture solution is a chondrocyte culture solution.
(2) Extracting mesenchymal stem cells, and inoculating the mesenchymal stem cells into an inserted cell culture dish; the extracted mesenchymal stem cells are primarily cultured mesenchymal stem cells. The inoculation amount of the mesenchymal stem cells is 3.5 multiplied by 104～4×104Per cm2。
(3) Putting the inserted cell culture dish in the step (2) and the chondrocytes cultured in the step (1) into a cell induced differentiation culture medium for culturing together; the culture is carried out in an incubator under the conditions of 37 ℃ and CO25 percent, pH value of 7.2-7.4, and sterile constant-temperature culture.
Wherein the cell induction differentiation culture medium takes a high-glucose DMEM culture solution as a basic culture medium, and also comprises the following components:
the concentration is 3.5X 10-7mol/L human bone morphogenetic protein 2;
the concentration is 4.8 × 10-7mol/L growth hormone HGH;
vitamin C at a concentration of 90 μ g/ml;
basic fibroblast growth factor (bFGF) with the concentration of 60 ng/ml;
transforming growth factor TGF-alpha with a concentration of 25 ng/ml;
transforming growth factor TGF-beta at a concentration of 24 ng/ml;
insulin at a concentration of 15 μ g/ml;
insulin growth factor IGF-1 at a concentration of 15 ng/ml;
bone morphogenetic protein-like BMPs at a concentration of 9 ng/ml;
fetal bovine serum or autologous serum at a concentration of 25 v/v%;
dexamethasone at a concentration of 50 nmol/L;
sodium pyruvate at a concentration of 2 w%;
interleukin IL-1 at a concentration of 5.5 ng/ml.
Inducing and differentiating positive expression type II collagen cells in the chondrocytes to carry out flow detection, wherein the positive expression rate of the type II collagen is 54.29% on the 5 th day of induction, and the positive expression rate of the type II collagen is 93.24% on the 8 th day of induction; and (3) measuring the immunofluorescence intensity: the ratio of the fluorescence intensity of the positively expressed type II collagen cells to the fluorescence intensity of DAPI is increased by 15.57% on the induction day 5 and 21.76% on the induction day 8; the number of the obtained cells was 1.246X 10, respectively, as measured by proliferation rate5(ii) a Secreted GAG content determination: at induction day 5, the secreted GAG content was 326.82 μ g each; at induction day 8, the secreted GAG content was 418.94 μ g each.
Example 2
A culture method for improving differentiation of mesenchymal stem cells to chondrocytes comprises the following steps:
(1) extracting chondrocytes, and inoculating the chondrocytes into a cell culture solution for culture; the extracted chondrocytes are subcultured chondrocytes. The inoculation amount of the chondrocytes is 0.3 multiplied by 104～0.8×104Per cm2. The chondrocytes are cultured in a cell culture solution for 2 days, wherein the cell culture solution is a chondrocyte culture solution.
(2) Extracting mesenchymal stem cells, and inoculating the mesenchymal stem cells into an inserted cell culture dish; the extracted mesenchymal stem cells are in primary culture mesenchymal stem cells. The inoculation amount of the mesenchymal stem cells is 3 multiplied by 104～4×104Per cm2。
(3) Putting the inserted cell culture dish in the step (2) and the chondrocytes cultured in the step (1) into a cell induced differentiation culture medium for culturing together; the culture is carried out in an incubator under the conditions of 37 ℃ and CO25 percent, pH value of 7.2-7.4, and sterile constant-temperature culture.
Wherein the cell induction differentiation culture medium takes a high-glucose DMEM culture solution as a basic culture medium, and also comprises the following components:
the concentration is 5X 10-7mol/L human bone morphogenetic protein 2;
the concentration is 4.5X 10-7mol/L growth hormone HGH;
vitamin C at a concentration of 150. mu.g/ml;
basic fibroblast growth factor (bFGF) with the concentration of 40 ng/ml;
transforming growth factor TGF-alpha with the concentration of 20 ng/ml;
transforming growth factor TGF-beta at a concentration of 15 ng/ml;
insulin at a concentration of 15 μ g/ml;
insulin growth factor IGF-1 at a concentration of 21 ng/ml;
bone morphogenetic protein-like BMPs with a concentration of 6 ng/ml;
fetal bovine serum or autologous serum at a concentration of 20 v/v%;
dexamethasone at a concentration of 60 nmol/L;
sodium pyruvate at a concentration of 1 w%;
interleukin IL-1 at a concentration of 7 ng/ml.
Also comprises tanshinone with concentration of 3mg/L, safflower powder with concentration of 0.8mg/L, total saponin of radix Achyranthis bidentatae with concentration of 1.5mg/L, and L-glutamine with concentration of 3 v/v%.
Carrying out flow detection on the positively expressed type II collagen cells induced and differentiated into the chondrocytes, wherein the positive expression rate of the type II collagen is 46.23% on the 5 th day of induction, and the positive expression rate of the type II collagen is 89.24% on the 8 th day of induction; and (3) measuring the immunofluorescence intensity: the ratio of the fluorescence intensity of the positively expressed type II collagen cells to the fluorescence intensity of DAPI is increased by 15.38% on the induction day 5 and 18.65% on the induction day 8; the number of the obtained cells was 1.286X 10, respectively, as a result of measurement of proliferation rate5(ii) a Secreted GAG content determination: at induction day 5, the secreted GAG content was 326.81 μ g each; at induction day 8, the secreted GAG content was 435.27 μ g each.
Example 3
A culture method for improving differentiation of mesenchymal stem cells to chondrocytes comprises the following steps:
(1) extracting chondrocytes, and inoculating the chondrocytes into a cell culture solution for culture; the extracted chondrocytes are two mixtures of primary cultured chondrocytes or subcultured chondrocytes. The inoculation amount of the chondrocytes is 0.3 multiplied by 104～0.8×104Per cm2. The chondrocytes are cultured in a cell culture solution for 1 day, wherein the cell culture solution is a chondrocyte culture solution.
(2) Extracting mesenchymal stem cells, and inoculating the mesenchymal stem cells into an inserted cell culture dish; the extracted mesenchymal stem cells are one or two of primary cultured mesenchymal stem cells. The inoculation amount of the mesenchymal stem cells is 3 multiplied by 104～5×104Per cm2。
(3) Putting the inserted cell culture dish in the step (2) and the chondrocytes cultured in the step (1) into a cell induced differentiation culture medium for culturing together; the culture is carried out in an incubator under the conditions of 37 ℃ and CO25 percent, pH value of 7.2-7.4, and sterile constant-temperature culture.
Wherein the cell induction differentiation culture medium takes a high-glucose DMEM culture solution as a basic culture medium, and also comprises the following components:
the concentration is 2X 10-7mol/L human bone morphogenetic protein 2;
the concentration is 4.8 × 10-7mol/L growth hormone HGH;
vitamin C with a concentration of 80 μ g/ml;
basic fibroblast growth factor (bFGF) with the concentration of 70 ng/ml;
transforming growth factor TGF-alpha with the concentration of 20 ng/ml;
transforming growth factor TGF-beta at a concentration of 15 ng/ml;
insulin at a concentration of 5.5 μ g/ml;
insulin growth factor IGF-1 at a concentration of 25 ng/ml;
bone morphogenetic protein-like BMPs at a concentration of 10 ng/ml;
fetal bovine serum or autologous serum at a concentration of 5 v/v%;
dexamethasone with a concentration of 70 nmol/L;
sodium pyruvate at a concentration of 1 w%;
interleukin IL-1 at a concentration of 15 ng/ml.
Inducing and differentiating positive expression type II collagen cells in the chondrocytes to carry out flow detection, wherein the positive expression rate of the type II collagen is 56.13% on the 5 th day of induction, and the positive expression rate of the type II collagen is 87.91% on the 8 th day of induction; and (3) measuring the immunofluorescence intensity: the ratio of the fluorescence intensity of the positively expressed type II collagen cells to the fluorescence intensity of DAPI is increased by 15.38% on the induction day 5 and 18.94% on the induction day 8; the number of the obtained cells was 1.652X 10, respectively, as a result of measurement of proliferation rate5(ii) a Secreted GAG content determination: at induction day 5, the secreted GAG content was 324.86 μ g each; at induction day 8, the secreted GAG content was 486.25 μ g each.
Example 4
A culture method for improving differentiation of mesenchymal stem cells to chondrocytes comprises the following steps:
(1) extracting chondrocytes, and inoculating the chondrocytes into a cell culture solution for culture; the extracted chondrocytes are subcultured chondrocytes. The inoculation amount of the chondrocytes is 0.3 multiplied by 104～0.8×104Per cm2. The chondrocytes are cultured in a cell culture solution for 2 days, wherein the cell culture solution is a chondrocyte culture solution.
(2) Extracting mesenchymal stem cells, and inoculating the mesenchymal stem cells into an inserted cell culture dish; the extracted mesenchymal stem cells are mixed in primary culture and subculture mesenchymal stem cells. The inoculation amount of the mesenchymal stem cells is 3 multiplied by 104～5×104Per cm2。
(3) Putting the inserted cell culture dish in the step (2) and the chondrocytes cultured in the step (1) together in a cell induction componentCulturing in a chemical culture medium; the culture is carried out in an incubator under the conditions of 37 ℃ and CO25 percent, pH value of 7.2-7.4, and sterile constant-temperature culture.
Wherein the cell induction differentiation culture medium takes a high-glucose DMEM culture solution as a basic culture medium, and also comprises the following components:
the concentration is 3.5X 10-7mol/L human bone morphogenetic protein 2;
the concentration is 5.6X 10-7mol/L growth hormone HGH;
vitamin C at a concentration of 120. mu.g/ml;
basic fibroblast growth factor (bFGF) with the concentration of 60 ng/ml;
transforming growth factor TGF-alpha at a concentration of 28 ng/ml;
transforming growth factor TGF-beta at a concentration of 25 ng/ml;
insulin at a concentration of 25 μ g/ml;
insulin growth factor IGF-1 at a concentration of 2 ng/ml;
bone morphogenetic protein-like BMPs at a concentration of 3 ng/ml;
fetal bovine serum or autologous serum at a concentration of 17 v/v%;
dexamethasone with a concentration of 70 nmol/L;
sodium pyruvate at a concentration of 1 w%;
interleukin IL-1 at a concentration of 12 ng/ml.
Also comprises tanshinone with concentration of 3mg/L, safflower powder with concentration of 0.50mg/L, total saponins of Achyranthis radix with concentration of 2.5mg/L, and L-glutamine with concentration of 3 v/v%.
Inducing and differentiating positive expression type II collagen cells in the chondrocytes to carry out flow detection, wherein the positive expression rate of the type II collagen is 61.43 percent on the 5 th day of induction, and the positive expression rate of the type II collagen is 92.48 percent on the 8 th day of induction; and (3) measuring the immunofluorescence intensity: the ratio of the fluorescence intensity of the positively expressed type II collagen cells to the fluorescence intensity of DAPI is improved by 27.24% on the induction day 5 and by 16.49% on the induction day 8; measurement of proliferation Rate: the number of the obtained cells was 1.5832X 10, respectively5(ii) a Secreted GAG content determination: in thatOn the 5 th day of induction, the secreted GAG content was 341.56 μ g; at induction day 8, the secreted GAG content was 453.28 μ g each.

Claims (7)

1. A culture method for improving differentiation of mesenchymal stem cells into chondrocytes is characterized by comprising the following steps:
(1) extracting chondrocytes, and inoculating the chondrocytes into a cell culture solution for culture;
(2) extracting mesenchymal stem cells, and inoculating the mesenchymal stem cells into an inserted cell culture dish;
(3) putting the inserted cell culture dish in the step (2) and the chondrocytes cultured in the step (1) into a cell induced differentiation culture medium for culturing together;
wherein the cell induction differentiation culture medium takes a high-glucose DMEM culture solution as a basic culture medium and comprises the following components:
the concentration is 2X 10-7mol/L human bone morphogenetic protein 2;
the concentration is 4.8 × 10-7mol/L growth hormone HGH;
vitamin C with a concentration of 80 μ g/ml;
basic fibroblast growth factor (bFGF) with the concentration of 70 ng/ml;
transforming growth factor TGF-alpha with the concentration of 20 ng/ml;
transforming growth factor TGF-beta at a concentration of 15 ng/ml;
insulin at a concentration of 5.5 μ g/ml;
insulin growth factor IGF-1 at a concentration of 25 ng/ml;
bone morphogenetic protein-like BMPs at a concentration of 10 ng/ml;
fetal bovine serum or autologous serum at a concentration of 5 v/v%;
dexamethasone with a concentration of 70 nmol/L;
sodium pyruvate at a concentration of 1 w%;
interleukin IL-1 at a concentration of 15 ng/ml.
2. The method of claim 1 for enhancing indirect chargingA method for culturing a mesenchymal stem cell into a chondrocyte, wherein the chondrocyte is inoculated in an amount of 0.3X 10 in step (1)4～0.8×104Per cm2。
3. The culture method for improving differentiation of mesenchymal stem cells into chondrocytes according to claim 1, wherein the chondrocytes in step (1) are cultured in a cell culture solution for 1-2 days, wherein the cell culture solution is a chondrocyte culture solution.
4. The culture method for improving differentiation of mesenchymal stem cells into chondrocytes according to claim 1, wherein the chondrocytes extracted in step (1) are chondrocytes obtained by one or a mixture of primary culture and subculture.
5. The culture method for improving differentiation of mesenchymal stem cells into chondrocytes according to claim 1, wherein the amount of the mesenchymal stem cells inoculated in step (2) is 3 x 104～5×104Per cm2。
6. The culture method for improving differentiation of mesenchymal stem cells into chondrocytes according to claim 1, wherein: the mesenchymal stem cells extracted in the step (2) are one or two of primary culture mesenchymal stem cells or subculture mesenchymal stem cells.
7. The culture method for improving differentiation of mesenchymal stem cells into chondrocytes according to claim 1, wherein in the step (3), the culture is performed in an incubator at 37 ℃ and CO25 percent, pH value of 7.2-7.4, and aseptic constant-temperature culture.
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